Association-dissociation phenomena in glycinin

Association-Dissociation

Iicceivcd

Phenomena

Jsll~~ar~- 6, 11Ki9; accepted

Febranry

in Glycinin

22, 1Wl

Ultraviolet, difference spectra itldicated that both tyrosille (287 mp) :rtld t ryptophan (293 mp) gror~ps are exposed by ~reatmc~rt of glycilGl with different coIIcentr;ttions of {Irea and with acid (pI1 2.0). (:lycinitl sollIt ions (O.:H”,) when heated at, temperat.ures above 70” become illcreasinglg turbid and the prot,eitl precipitates at ‘JO”. (;lycinin appears to he stable to heating 11p to 50” and very lit tie change is observed between 50 and TO”. IIeat, acid (~11 2.0), alkali (pII ll.O), :ttld [Irea treatment cause dissociation of glycinin illto srlbrlllits atld subunit association prod\Lcts as esamined by disc electrophoresis. Undissociated glycinin molecrdes call be detected after heat, acid, and alkali treatment by double gel immrllrodiff~lsioll RII~ irnmrlnoelect~rophoresis. Urea (6 M) causes irreversible and complete dissociation of the protein illto sllbunits. The sllbrrnits of glycillin, and reduced and carbox;vmcth?lated glycillin (I:CRL-glgciIAn) are soluble in the solvent system phclrolLcetic acid-O.2 \I mercaptoethallol, 2:1:1, w/v/v made 5 ix in llren (PAXlU). 1)isc electrophoresis llsillg the PAMU solvellt, alld five major sllbmlits from separates six major sub-IuAts derived from glycinitl, KCM-glycillitl. (;elleral cotlsideratiotls from exist)itlg data aborlt the strrlct ,lre of . glycinin are discllsscd. Glycininl

is the

major

reserve

protein

data, represent dissociated subunits, monomer, dimer, and polymers of this protein, respectively. The conversion of glycinin into a 2s component has been shown to be irreversible (13). Similarly, dissociation of glycinin into subunit’s has been shown in this work to be irreversible. The other forms may be interconvertible (9-1X). The biological role of glycinin in the germinating soybean seed appears to be that of a storage protein which is degraded during the first’ 1G days of germination (1, 15). It has been suggested in previous ivork (15) that glgcinin is dissociat’ed into subunits before final utilization by the seedling. The proposed mechanism is supported also by the \\-ork of I’ukushima (16) who demonstrated the glycinin is not hydrolyzed by proteinases without disruption of its tertiary structure. Thus, study of associatiowdissociation pheuomena in glycinin mav contribute to a better understanding ok the degradation process of storage proteins in the seedling and to improved utilization of these proteins in humnn nutrition (17).

of

soybean seeds (Glycine max) located in subcellular particles called cotyledonary “protein bodies” (l-:3). This protein has been purified by DEAE-Sephadex chromatography (4) and shown to be homogeneous by disc electrophoresis and by several immunochemical methods (5, G). As judged by ultracentrifugation, glycinin has also been purified by other chromatogrtphic methods (T-9). The S-terminal amino acids of glycinin are glvcine, phenylalaninc, and leucine or isolekine (4, 7) in the molar ratio of 4: 1: 1, respectively- (4). The molecular weight of glycinin has been estimated to be 350,000 (10). The protein forms disulfide polymers (10, 11) and is dissociated into subunits under different cxperimental conditions (4, 12-14). The four ultracentrifugal forms of glycinin (9-13) expressed by the scheme: 2s + 7s s 11s e 15S, to the best judgment based on existing

5TT

575

CATSIMPOOLAS,

CAMPBELL.

This paper describes pH, temperature, and urea effects on the physical structure of glycinin as examined by disc electrophoresis, ultraviolet difference spectra, and immunochemical methods. EXPERIMENTBL

PROCEDUHE

Preparation of glycinin samples. Glycinin was prepared as described previously (4). For the experiments involving heat denaturation, the protein was dissolved in pH 7.G phosphate buffer (0.0235 M KzHPOa , 0.0026 M KH,POa) made 0.4 M in sodium chloride and 0.01 M in mercaptoethanol. This has been designated as standard buffer (10). The protein solutions (3.4 mg/ml) were heat,ed in a water bath at t,he appropriate temperat,ure for 1 hr. The heated solutions were left to cool slowly to room temperature. These samples were then used for disc electrophoresis and immunodiffusion analyses. It was noticed that the protein solution became cloudy at 80” and a precipitate was formed by heating at 90”. These two samples were centrifuged and only the supernate was used for analysis. The rest of the solut’ions were clear. In order to study irreversible changes of glycinin due to the pH effect), the protein was dissolved in standard buffer (3.4 me/ml) and subjected to dialysis against large volumes of buffers at various pH values for 24 hr. After this period, the treated samples were redialyzed against several changes of the standard buffer for a period of 72 hr. All the buffers were made 0.4 M in NaCl to eliminate the possibility of changes due to differences in ionic strength. Reduced and carboxymethylated glycinin (RCM2-glycinin) was prepared by the general procedure of Crestfield et al. (18) with the only exception that reduction with mercaptoethanol was carried out in 6 M urea solution. Disc electrophoresis. Ordinary disc electrophoresis on 79;;! polyacrylamide gel columns (with and without incorporation of Ilrea) was carried out by the general procedure of l>avis (19). Disc electrophoresis using the PAiL’IU? solvent (phenolacetic acid-O.2 M mercaptoethanol, 2: 1: 1, w/v/v made 5 M in urea) was performed as described previously (15). Immunochemical methods. Antisera to soybean proteins were prepared by immrlnization procedures described previorlsly (2, 5, 6). The experimental conditions for double gel immunodiffllsion and immunoelectrophoretic analysis of soybean proteins have been reported (2, 6). 2 Abbreviations boxymethylated; mercaptoethanol,

used: RCJI, reduced and carPARIU, phenol-acetic acid-O.2 M 2: 1: 1, w/v/v made 5 M in Ilrea.

BND

MEYER

Ultrauiolel diflerence spectra. Difference spectra were obtained using a Beckman DU spectrophotometer equipped with a photomultiplier attachment and l-cm quartz cuvettes. Temperature control of the sample was maintained by the use of thermospacers and a Haake constant temperature circulator. When applicable, temperature changes were recorded by using a glass thermistor probe attached to a YSI telethermometer. The measuremerlts were made using a 0.34Ljl protein solution. Differences in absorbance (-AA) at each wavelength were obtained by adjusting the spectrophotometer to zero absorbance against the experiment,al protein sample and t’hen reading the absorbance of the reference glycinin solrltion in standard bluffer. When temperat,ure-induced aggregation of glycitlin was recorded, the heated glycinin sollltioll was used for reading the absorbance and the unheated glycinin solution served as reference. RESULTS

AND

DISCUSSION

L’ltraviolet difference spectra. The effect of different concentrations of urea on the ultraviolet difference spectra of glycinin is shown in Fig. 1. Four maximum negative peaks were obtained at 245, 280, 257, and 293 rnp upon exposure of glycinin to urea solutions for 30 min. The change in absorbancy at 280 and 257 rnp can be assigned to the exposure of tyrosine, and that at 293 rnp to tryptophan side groups (20). Thus, it appears that both tyrosine and tryptophan are buried in hydrophobic regions in the native glycinin molecule. Exposure of these groups by the action of urea on the native molecule may be the result of (a) dissociation into subunits, i,b) unfolding or swelling of each compact subunit structure, and (c) a combination of (a) and (b). Figure 2 shows the changes in absorbance at 287 and 293 rnp as a function of urea concentration. As the concentration of urea increases the characteristic “denaturation blue shift” (21) is observed, reaching a maximum when the urea concentration is near 6 M. It is apparently in this region of urea concentration that glycinin undergoes a profound conformational change. At S M concentration of urea a red shift occurs which is probably due to the increase in the refractive index of the solvent (20). It may be seen that exposure of tyrosine (“ST mp) and tryptophun (293 mp) groups at different con

(:LYCININ

rlSSOCIhTION-DISSOCIrlTION

PIIENOXEN.4

-AA

WAVELENGIW

(mp)

in different conFIG. 1. Ultraviolet difference spectra of glycinin (0.34~~ concent,ration) centrations of Ilrea. Key: 2 M urea (A), 4 M urea (01, (i Y wea (0), and 8 M urea (0).

centrations of urea run almost in parallel. Thus, preferential exposure of either one of these groups at a certain concentration of urea does not appear to take place. The peak with a maximum at approximately 245 rnp has not been assigned to any particular group. This peak \vas also observed when glycinin was exposed to pH 2.0 for 30 min (Fig. 3). Thus, spectral changes in this ultraviolet region occur n-hen glycinin is dissociated in either urea or acid. Wootton and Hess (22) have demonstrated with Lu-chymotrypsin catalyzed reactions that absorbancy changes in the 245rnp region are due to light scattering resulting from molecular aggregation. In our experiments, when the glycinin concentr:r-

tion was lowered from 0.34% to 0.0,5% the absorbance maximum at 245 rnp was shifted to 23s rnp. This is an indication that at the higher protein concentration the 245 rnp peak is probably due to molecular aggregation of the dissociated glgcinin subunits. Bukushima (16) also reported that at wavelengths below 2’50 mp, glgcinin concentrations of 0.04% were employed to eliminate stray light effects. Similarly, a peak at 235 rnp was demonstrated. In an attempt to follow the exposure of tyrosine groups (“87 mp) during thermal denaturation of glycinin, it was observed that a positive absorbance difference was obtained instead of the usual negative change seen with urea and acid. This change was

580

CATSIRlPWLAS,

CAJIPBELL,

SKI)

JIEYEI~

no\v that the protein appears to be quite stable to heat treatment. When heated at temperatures above 70”, dissociation of the protein into subunits occurs, apparently followed by aggregation either of the subunits or of a combination of subunits and partially rearranged glycinin molecules. Disc electrophoresis. Freshly prepared gly287 mp cinin in standard buffer (4) exhibits one band ivhen examined by disc electrophoresis as shown in Fig. 3A. However, the protein on storage, freeze-drying, isoelectric precipitation, change in ionic strength of the I I / I buffer, and on mild heat treatment exhibits the pattern showy in Fig. 5B. Tombs (3) has assigned these bands to represent the monomer (m), dimer (d), and polymers (p) of glycinin. When these components are allowed to diffuse either against anti-glycinin serum or anti-whole soybean extract serum only one immunoprecipitin arc is formed as / 2 4 6 6 0 examined by the technique of disc immunoUREA CM, electrophoresis (1). Thus, the three strucFIG. 2. Ultraviolet difference spectra of glytural forms of glycinin (namely monomer, chin (0.347, concentration) at 287 mp (A) and dimer, and polymers) are immunochemically 293 mp (0) as a function of urea concentration. identical. When the total reserve proteins of the soybean (1) are examined by disc elecfound to be the result of turbidity developed trophoresis two other protein components, in the heated glycinin sample and not a namely beta and gamma conglycinins (23), specific absorbance phenomenon. Apparently migrate at approximately the same position it can be measured at other wavelengths. as the monomer of glycinin and can be The protein concentration was 0.34% in distinguished from the latter only by the standard buffer. The rate of heating of the difference in immunochemical specificity (1, sample at different temperatures and ab- 23). sorbance changes at 287 rnp are shown in From disc electrophoresis (present report), Fig. 4. No significant changes were observed polyacrylamide gel electrophoresis (3)) and by heating glycinin up to 50”. Examination ultracentrifugal data (12, 13), it appears that of samples heated at these temperatures bJ the most abundant form of glycinin is the disc electrophoresis did not show the pres- dimer which exhibits a sedimentation coence of subunits. Very little change is ob- efficient of 11s. The monomer, referred to in served between 50” and 70”. However, a the past as “half-molecule” of the 11s soysignificant and sudden change occurs when bean protein, has a sedimentation coefficient the protein is heated at temperatures above of 7s (12, 13), and the polymers, 15s and 70”. Glycinin is partially precipitated when higher. That the dimer of glycinin (11s) is heated at 90”. These samples (heated at dissociated into two identical 7s half-mole70-90”)) when examined by disc electrocules (monomers) is supported by the followphoresis, exhibited significant dissociation of ing data: (a) an even number of polypeptide the protein into subunits (e.g. Fig. 5E). chains having the same K-terminal amino However, undissociated glycinin could be acid (eight glycine, two phenylalanine, and detected by both disc electrophoresis and two leucine or isoleucine residues per 350,000 immunochemical methods (see below). Since g protein) exist in glycinin (4) ; and (b) thermal denaturation of glycinin deserves the dimer and monomer are immunochemimore extensive studies, it will suflicc to sa!- tally identical. If glycinin were dissociated

WAVELENGTH (mr) FIG.

3.

TJltrnviolet

diflcre~rce specie

of glycillill

into two different half-molecules, e.g. A and R, then the purified dimer exhibiting initially one immunoprecipitin arc, should after partial dissocation (heat, pH change, etc.) give three immunoprecipitin arcs: t’hosc of undissociated glycinin, half-molecule A, and half-molecule H, when developed with anti-whole soybean extract serum. As will be discussed later, only one immunoprecipitin arc is detected by double gel diffusion and immunoelectrophorcsis. It is of course assumed that if forms A and B existed, these should have been suficiently antigenic to elicit antibody response. When glycinin is exposed to highly acidic and alkaline buffers (e.g. pH 2 and pH 11) and then redialyzed against the pH 7.6 standard buffer, irreversible dissociation into subwits cm be dct’ectcd b>- disc elect’rophoresis. Figures SC and SD she\\- the disc clectrophoresis pat’tern of glycinin exposed to pH 2 and pH 11 (SW L\Iethods), The relative mobility and respectively. number of subunits produced in each case is different. This leads us to assume that these

(O.;H(~, cotlrerltriltiull)

at

prr 2.0

subunits may reprcscnt subunit-associ:Ltit,rl products and not. single chain subunits (at least not all of them). Undissocint’ed glycinin was also found to be present as seer1by disc electrophoresis and by immunochen-kal methods (see below\-). The presence of undissocintcd glycinin does not appear to be the result of reversible associxt’ion of the subunits but rather of incomplete dissociation of glgcinin. This assumption \vas confirmed by exposing glycinin to (5 .\I urea and then redialyzing against the pH 7.6 standard buffer. LJndissociated glycinin molecules could not be detected either by double gel immunodiffusion or immunoclectrophorcsis. The disc electrophoresis pattern (Fig. 51:) shows subunits and apparently aggregation products that remain at the origin of the gel. A subunit which appears to migrate between the position of the monomer and dimer of glycinin did not form an immunoprecipitin arc by disc immunoelectrophoresis. Thus, it can be concluded t’hat \vhen glycinin is complet’ely dissociated, as in t,hc presence of 6 .\I urea, the molecule

582

FIG. 1. Rleasurement diferent temperatures

(at 287 mr) of turbidity developed by heatittg (0). The rate of heatittg is indicated (0).

glycittitt

solutions

at

FIG. 5. Regular disc electrophoresis analysis of freshly prepared glyciuin in standard buffer (A), glycinin after storage or mild treatment (see t,ext) (IS), glycittin exposed to pH 2.0 (C), pH 11.0 (D) and redialyzed against the standard buffer, superttatant of glycinirt heat,ed at 90” (E), and glycittin dissociated itt 6 M urea aud redialyzed against the standard buffer (F). Glycinin monomer (m), dimcr (d), attd polymers (p).

cannot be reformed. The dissociation into subunits must then bcb an irreversible process. In cases where undissociated glycinin can be d&c&d, it is because of incomplete dissociation rather than reversible association of subunits. When glj.cinin is dissociated in ci :\I urea containing 0.2 31 mercaptoethanol or 6 I\1 guanidine hydrochloride containing 0.2 :\I and then subjected to mercaptoethanol, disc electrophoresis in gels containing urea (4), a number of subunit’s can be seen, but also material that remains at the origin of the gel is present (l’ig. GH). Under these ap it becomes experimental conditions,

parent that glJG]in subunits undergo ussociation reactions and assignmcnt~ of single chain subunits is very diflkk However, dissociation of glycinin in I’hl\ZU solvent follon-ed by disc clectrophoresis in the same solvent (15) offers more meaningful data, since the total dissociated protein material is soluble in this solvent, and migrates in the polyacrylamide gel without streaking or material left at’ the origin. Incorporation of mercaptoethanol has been found to be necessary in order to achieve a more uniform pattern of subunits (15). Glycinin dissociat’ed in I’AMU solvent cshibits six major bxnds I\-hich account for

584

CATSI-\IPOOLSS.

CBMPBELL,

92% of the total densitometer tracing area (Fig. GA). Six ot(her minor bands account for the remaining S ‘i; of the densitometer tracing area. The minor bands (since each one represents l-2% of the tracing only) may be either contaminants attached to the protein, or minor association products of the subunits. Reduced and carboxy= methylated glycinin dissolved in 1’,41\lU solvent and examined by disc electrophoresis in the same solvent shows live major bands and traces of two others (Fig. 7). Four of these major bands appear to migrate in pairs. The fastest moving band migrating towards the negative electrode exhibits higher intensity than each of the other bands, as indicated by the microdensitometer tracing, and may represent two components with identical relative electrophoretic mobilities. In any case, the conclusion from the disc electrophoresis experiments in PAJIU solvent is that glycinin is dissociated into five or six major subunits. At present, it is not known whether these components represent single chain subunits or one or more of the subunits are overlapping because of similar relative electrophoretic mobility. Isolation of the subunits in sufficient quantity and individual N-terminal amino acid analysis will be necessary for positive identification. How

FIG. 7. Disc electrophoresis &in. The Inicrodensit.ometrr tometer.

ANI)

1\[EYE:R

ever, the existence of six single chain subunits will be more consistent with N-terminal amino acid data of undissociated glycinin (4), with the hypothesis based on immunochemical data of two identical monomers, and with the expectation that a symmetrical molecule is more stable and therefore more probable. It should be mentioned that arachin, a protein closely related to glycinin, also consists of two monomers each conzposed of six subunits (24). Inlr,lunochellzistl.y. Glycinin dissolved in standard buffer and heated at different temperatures for 1 hour exhibits one immunoprecipitin band when examined by double gel diffusion using anti-whole soybean extract serum (Fig. S). Since n-e have shown that glycinin dissociated completely into subunits (e.g. 6 XI urea treatment and removal of urea) does not give an immunochemical reaction with antiserum, it can be assumed that the immunoprecipitin band obtained is due to undissociated glycinin. It appears that glycinin is not entirely destroyed even by heating up to 90”. How ever, significant precipitation of the protein occurs by heating at 90” and above. Prolonged heating will eventually destroy the protein at these temperatures. Glycinin exposed to pH values ranging from pH 2 to pH 11 and redialyzed against

in PAhlU solvent of reduced and carboxymethylated glytracing was obtained with a Canalco model F microdensi-

&eat~inetrt

al, differeJt”

JIJ~

vallm

(set test).

t#hc pH ‘7.6 standard buffer also exhibits one immunoprecipitin arc by double gel diffusion (I’ig. 8). Again undissociuted glycinin ma? account for this renctivit’y. The most’ significant finding of these cxperiments is that’ glycinin under different condition of pH and heat treatment does not dissociate int’o components immunochemically different from the parent molecule. Thus, the isolated alpha, beta, and gamma conglycinins (23) :are not brc:rkdo\vn products of glycinin formed during isoLtion procedures, but individual proteins. This has been confirmed by analysis of the subunit composition of the conglycinins (unpublished results). Immunoelcctrophoretic analyses of glycinin, subjected t’o identical treatment as in the described double gel diffusion experiments, showed only one immunoprecipitin ax of approximately the same electrophoretic mobility. This is ZLI~ additional indication that the immunochemical reactivity under denaturing conditions is due to undissociated glycinin molecules ivhich wwe also sho~~-~~ t,o be present by disc elcctrophoresis. Further ~~)rk on the isolation of the subunits of glycinin and stud.y of their interactions may contribute to a better understanding of the forces responsible for the stabilization of complex protein molecules in general and more specifically of the role of the storage proteins in the germinating seedling. With the increase use of soybean

proteins in animal and human nutrition such studies may :11so lead to improved utilization.

5.

C.4~~sl~~l~ooI,as,

K.

(i.

~~A~L’HlMl’OOl,AS,

Hiochem. i.

8.

x.

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H., Eil-S,lSO, K.. dgr. B’iol. Chm.

~IiVAlt’l‘S,

J’;LI)I~II>GE,

(‘hew.

~IE:YEIL,

I’.

\v.,

./.

.t!/r.

\..

s.

.4SU

(T~rSSI~) i\.

c.

hXEl1,

1,;. iv..

.d

rch.

126, 742 (1968).

Riophys.

~JITSI-Ij.4,

chrr)ci.s/r!/ ‘1

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16, 128 (lW3).

F’ootl (‘hem.

1’.,

.4SU

JJASEGA\\.A,

(‘l’Ok!/O) 29, i (1965,. \~.AINrl’llAl-l~,

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h.,

HII,-

32, lZ5 (1967). ANI)

44, 615 (1W).

b\‘OLF,

k’.

I.,

(‘c,wu~

5M

CATHIMPOOLAS,

CAMPBELL,

lfi. FUKUSHIMA, l)., Cereal Chem. 46, 203 (1968). 17. ALTSCHUL, A. RI., Science 168, 221 (19G7). 18. CRESTFIELD, A. iXI., ~IOOI~E, S., AND STEIN, W. H., J. Hiol. Chern. 238, (i22 (19G3). 19. I)AVIS, B. J., Ann. .\‘. I’. dcatl. Sci. 121, 404 (1964). 20. I~DSALL, J. T., In “Aspects of Prot,eitl Struttnre” (G. N. l
ANI)

RIEYEI:

21. BIGELO\V, C. C., C. R. Trav. Lab. Carlsbery 31, 305 (19CO). 22. \YOOTTON, J. F. AND HESS, (;. P., J. Am. Chem. Sot. 83, 4232 (19Cil).

23. ~ATSIMPOOI,AS, 5. ANI) ~';KENsTAM, C., Arch. Riochem.

Nioph,yw. 129, -290 (19G9).

2-L To~llrts, Il. P. AND lJ~)~~~~~, AI., Uiochem. 106, 181 (19li7).

.J.